The present invention relates to transgenic plants presenting a modified inulin producing profile, to a method for producing said plants, to a method for modifying and controlling the inulin producing profile of plants and to a method for producing inulin from said transgenic plants.
Furthermore, the present invention relates to novel 1-SST and 1-FFT enzyme encoding DNA sequences, to novel recombinant DNA constructs and recombinant genes derived thereof, to novel combinations of expressible 1-SST and 1-FFT enzyme encoding genes, as well as to novel polypeptides or fragments thereof presenting 1-SST and/or 1-FFT activity, and to antibodies capable of binding to them.
Inulin is a fructan type carbohydrate polymer which occurs as a polydisperse composition in many plants and can also be produced by certain bacteria and fungi. Inulin from plant origin consists of a polydisperse composition of mainly linear chains composed of fructose units, mostly terminating in one glucose unit, which are linked to each other through xcex2(2-1) fructosyl-fructose linkages.
Inulin can be generally represented, depending from the terminal carbohydrate unit, by the formulae GFn and Fm, wherein G and F respectively represent a glucose unit and a fructose unit, n is an integer representing the number of fructose units linked to the terminal glucose unit, and m is an integer representing the number of fructose units linked to each other in the polyfructose chain.
The number of saccharide units (fructose and glucose units) in one molecule, i.e. the values n+1 and m in the above formulae, are commonly referred to as the degree of polymerisation, represented by DP. Often also the parameter (number) average degree of polymerisation, represented by ({overscore (DP)}) is used, which is the value {overscore (DP)}n calculated, after complete hydrolysis and considering that in native inulins the Fm fraction is negligible, as follows:             DP      _        n    =                    total        ⁢                  xe2x80x83                ⁢        %        ⁢                  xe2x80x83                ⁢        F                    total        ⁢                  xe2x80x83                ⁢        %        ⁢                  xe2x80x83                ⁢        G              +    1  
In the equation % refers to weight percent (wt %). Furthermore, in this calculation the saccharides glucose (G), fructose (F) and saccharose (GF) which are present in the polydisperse polysaccharide, should not be taken into account. The average degree of polymerisation is thus the ({overscore (DP)}n) of inulin, herein interchangeably referred to in short as ({overscore (DP)}) inulin or ({overscore (DP)}) (De Leenheer, 1996).
The polysaccharide chains of native inulin from plant origin generally have a degree of polymerisation (DP) ranging from 3 to about 100, whereas the ({overscore (DP)}) of the native inulin largely depends from the plant source, the growth phase of the plant, the harvesting time and the storage conditions. The ({overscore (DP)}) of isolated inulin largely depends on the ({overscore (DP)}) of the native inulin and on the process conditions used for the extraction, purification and isolation of the inulin from the plant or plant parts.
By native inulin or crude inulin is meant herein inulin that has been extracted from plants or parts of plants, without applying any process to increase or decrease the ({overscore (DP)}), while taking precautions to inhibit the plant""s own hydrolase activity and to avoid hydrolysis. The ({overscore (DP)}) of native inulin thus essentially corresponds to the ({overscore (DP)}) of the inulin as present in the plant or plant parts.
The isolated inulin obtained from plants or plant parts through conventional manufacturing techniques, commonly including extraction, purification and isolation, without any process to modify the ({overscore (DP)}) of the native inulin, is termed herein, interchangeably, standard ({overscore (DP)}) grade inulin or standard grade inulin. As a consequence of the manufacturing process, the ({overscore (DP)}) of standard grade inulin is usually about 1 to 1.5 lower than the ({overscore (DP)}) of the native inulin.
Inulin molecules with a DPxe2x89xa610 are commonly termed oligofructose, inulo-oligosaccharides or fructo-oligosaccharides (in short FOS). Both, inulin chains with a DPxe2x89xa610 and inulin chains with a DP greater than 10, are embraced herein by the term inulin.
By inulin profile is meant the relative composition of the polydisperse inulin as formed by the individual components including glucose, fructose, sucrose and individual inulin chains, including the distribution pattern of the polyfructose (inulin) chains.
Linear inulin is common in a specific plant family, the Asteraceae, including the plant species Jerusalem artichoke (Helianthus tuberosus) and chicory (Cichorium intybus). Inulin is commonly stored in tap roots (chicory) or in tubers (Jerusalem artichoke) and acts as a storage reserve for regrowth of the sprout after the winter period.
Accordingly, typical sources for the production of inulin at industrial scale are roots of chicory and, on a much smaller scale, tubers of Jerusalem artichoke, in which inulin can be present in concentrations of about 14% to 18% by weight on fresh weight. Inulin can be readily extracted from these plant parts, purified and optionally fractionated in order to remove impurities, monosaccharides, disaccharides and undesired oligosaccharides, as for example described in PCT patent application WO 96/01849.
Conventional processing of roots of chicory yields a standard grade inulin, containing about 8 wt % of mono- and di-saccharides (including glucose, fructose and sucrose) and a polydisperse mixture of inulin molecules with a DP ranging from 3 to about 60 and a ({overscore (DP)}) of about 10. The DP of the inulin molecules of standard grade inulin from Jerusalem Artichoke tubers ranges from 3 to about 40 whereas the ({overscore (DP)}) is about 7.
It is known that in Asteraceaous plants, including Jerusalem artichoke and chicory, inulin molecules are synthesised by the concerted action of two enzymes:sucrose:sucrose 1-fructosyltransferase (in short 1-SST enzyme or 1-SST, used interchangeably) and fructan:fructan 1-fructosyltransferase (in short 1-FFT enzyme or 1-FFT, used interchangeably) (Koops and Jonker, 1994 and 1996). Both 1-SST and 1-FFT are active during the period of inulin synthesis and accumulation:
1-SST catalyses the initial reaction of inulin biosynthesis, the conversion of sucrose into the smallest inulin molecule, the trisaccharide kestose (GFF), according to:
GF+GFxe2x86x92GFF+Gxe2x80x83xe2x80x83(1)
1-FFT catalyses the redistribution of terminal fructosyl units (xe2x88x92F) between inulin molecules, which results in a stepwise increase in chain length, according to:
GFFn+GFFmxe2x86x92GFFnxe2x88x921+GFFm+1,xe2x80x83xe2x80x83(2)
(wherein n and m are integers  greater than 0)
Some examples of this type of reaction are
GFF+GFFxe2x86x92GFFF+GFxe2x80x83xe2x80x83(2a)
GFFF+GFFFxe2x86x92GFFFF+GFFxe2x80x83xe2x80x83(2b)
GFFFF+GFFFFxe2x86x92GFFFFF+GFFFxe2x80x83xe2x80x83(2c)
An essential difference between the 1-SST enzyme and the 1-FFT enzyme is that the 1-FFT enzyme cannot catalyse reaction (1). In contrast, the 1-SST enzyme, next to reaction (1), can catalyse reactions of type (2) yielding inulin molecules with a low DP (catalysis by known 1-SST enzymes being able to yield inulin molecules with a DP up to about 5).
Accordingly, in plants both the 1-SST and the 1-FFT enzymes are contributing to inulin synthesis and the profile of native inulin is determined, inter alia, by sucrose supply, expression of the 1-SST enzyme encoding genes and 1-FFT enzyme encoding genes and the kinetic properties and relative activity of the 1-SST and 1-FFT enzymes which may be controlled by the relative expression of the 1-SST and the 1-FFT enzyme encoding genes.
Inulin is an edible, water soluble polydisperse polysaccharide composition which is used in the manufacture of many food and feed products, drinks and non-food products. In food, feed and drinks, inulin can be used, inter alia, as a bulking agent as well as a total or partial substitute for sugar and/or fat. Furthermore, inulin can be added to food, feed and drinks to enrich them with soluble fibres having prebiotic properties. Moreover, inulin can also be used as a component of prophylactic and therapeutic compositions. Besides, inulin with a ({overscore (DP)}) of about 10 and more is commonly used at industrial scale as starting material for the manufacture of oligofructose and of fructose, which both are increasingly used in industry as sweeteners, particularly in drinks and fruit compositions.
Usually different applications require inulin with a different profile. For example for use as (oligofructose) sweetener, the inulin molecules should have a low DP, preferably about 3 to 8, whereas for use as fat replacer inulin should preferably have a ({overscore (DP)}) higher than 15. For non-food applications inulin may have to be derivatised, for example to obtain carboxymethylated inulin which can be used as sequestering agent for divalent cations. Inulin suitable as starting material in derivatisation reactions should preferably have a ({overscore (DP)}) of at least 20, whereas its level of low molecular weight sugars should be very low. Standard grade inulin from chicory or Jerusalem artichoke has a too low ({overscore (DP)}) and a too high level of low molecular weight sugars, which makes derivatisation of said inulins difficult.
To prepare inulin which is low in mono- and disaccharides and has a high average degree of polymerisation, preferably a ({overscore (DP)}) of at least 20, various techniques have already been disclosed, for example, a method of manufacture involving a directed crystallisation starting from native or standard grade chicory inulin as described in PCT patent application WO 96/01849. Inulin with such a profile is also very suitable as ingredient in various food, feed, drinks and non-food applications, and as starting material for the manufacture of hydrolysates and derivatives of inulin.
To prepare oligofructose, usually standard grade chicory inulin or preferably chicory inulin with a higher ({overscore (DP)}), e.g. a ({overscore (DP)}) of at least 20, is subjected to partial, enzymatic hydrolysis, whereas to prepare fructose, typically in the form of a fructose syrup, said inulins are subjected to complete enzymatic or acidic hydrolysis, as for example described in patent applications PCT/BE97/00087 and EP 97870111.8.
However, every treatment of native inulin or standard grade inulin to reduce the content of low molecular weight sugars, to increase the ({overscore (DP)}) of the inulin, to modify the inulin profile, particularly to modify the distribution pattern of the inulin chains of the source inulin, or to transform inulin into oligofructose, requires one or more additional processing steps, such as, for example, size fractioning or hydrolysis. These additional process steps inevitably result in technical and economical disadvantages.
Accordingly, in the search for methods for producing inulin with a predetermined profile also an other approach is being prospected which envisages the direct production of inulin with a desired profile from genetically modified plants showing a modified inulin producing profile.
Herein the terms genetically modified, transformed and transgenic are used interchangeably; the terms 1-SST or 1-SST enzyme and 1-FFT or 1-FFT enzyme refer to the respective enzymes, whereas the terms sst103 or sst103 sequence and fft111 or fft111 sequence indicate an example of a 1-SST, respectively 1-FFT encoding DNA sequence, and the terms sst103 gene and fft111 gene indicate an example of a 1-SST, respectively a 1-FFT enzyme encoding gene.
PCT patent application WO 96/01904 claims a method for producing fructo-oligosaccharides from a transgenic plant containing a gene construct comprising a fructosyl transferase encoding ftf gene from Streptococcus mutans or a fructosyl transferase encoding Sac B gene from Bacillus subtilus or a mutated version of said genes. The invention aims particularly low molecular fructo-oligo-saccharides having a DP 3 to 4.
The patent application WO 96/01904 describes the isolation of an SST enzyme from onion and shows the activity of the purified enzyme in vitro by incubation with sucrose with the formation of 1-kestose only. Neither the DNA sequence coding for this SST enzyme nor the amino acid sequence of the purified SST enzyme were disclosed. The patent application also discloses the sequences of two other fructosyl-transferases and the use of the 6-sft gene isolated from barley, where it is involved in the biosynthesis of non-inulin type branched fructans, in an heterologous screening leading to the isolation of the cDNA sequences of two virtually identical genes from the flowers of onion, respectively pAC22 and pAC92, which have been tested separately in protoplasts of tobacco. In the patent application (p. 19, line 27 to p. 20, line 2) it is indicated that in this way a fructosyltransferase activity could be shown but no experimental data were presented. Later experiments (Vijn et al., 1997) have revealed that pAC22 (designated pAC2 in Vijn et al., 1997) in fact encodes a 6-FFT enzyme (EMBL accession No Y07838) (fructan:fructan 6G-fructosyl transferase), which catalyses the transfer of a fructosyl residue to the carbon 6 of the glucose moiety of sucrose, resulting in the formation of the trisaccharide neokestose (F2-6G1-2F) according to: GFF+GFxe2x86x92FGF+GF, so that, although presenting fructosyltransferase activity, the disclosed sequences of pAC22 and pAC92 thus do not represent 1-SST coding sequences.
On the one hand there is an essential difference between a 6-G-FFT enzyme and a 1-SST enzyme since (i) the 6-G-FFT enzyme can not use saccharose as donor of a fructosyl residue (as a 1-SST enzyme does) but needs kestose as a fructosyl unit donor, and (ii) the 6-G-FFT enzyme catalyses the synthesis of neokestose (FGF) constituting the starting moiety for the building up of a fructan of the inulin neoseries class, whereas the 1-SST enzyme catalyses the synthesis of kestose (GFF) constituting the starting moiety for the building up of a fructan of the inulin class.
On the other hand there is an essential difference too between a 6-G-FFT enzyme and a 1-FFT enzyme, since the 6-G-FFT enzyme can catalyse only the production of polysaccharide chains with a low DP, i. e. a DPxe2x89xa6about 10, whereas a 1-FFT enzyme can catalyse the synthesis of polysaccharide chains with a higher DP, i.e. a DP up to about 70 and even up to about 100.
Said difference between the 6-G-FFT enzyme and the 1-SST enzyme, respectively the 1-FFT enzyme, is also reflected in the polysaccharide molecules built up through catalysis by said enzymes. The polysaccharide chains built up via a 6G-FFT enzyme catalysis are of the inulin neoseries class, reach a DP of only up to about 10, have not a terminal glucose moiety, and are not strictly linear as a result of the 16 disubstituted glucose moiety in the molecule, whereas the polysaccharide molecules built up via 1-SST enzyme and 1-FFT enzyme catalysis can reach a DP of up to about 70, even up to about 100, have a terminal glucose moiety, and present an essentially linear structure. Furthermore, the use of the individual sequences in different DNA constructs to produce transgenic plants, in particular different crops, is mentioned but no experimental data on the oligosaccharides obtained were given. Furthermore, Vijn et al., 1997 disclosed that introduction of the onion 6-G-FFT enzyme encoding sequence in chicory resulted in a transgenic plant which made linear inulins (i.e. genuine chicory inulin) and in addition fructans of the inulin neoseries. Apparently the native 1-SST enzyme encoding sequences together with the native 1-FFT enzyme encoding sequences of the chicory ensured via the corresponding enzymes the synthesis of the native inulin molecules, whereas the native 1-SST enzyme encoding sequences together with the onion 6G-FFT enzyme encoding sequence lead via the corresponding enzymes to the synthesis of inulin neoseries of low molecular weight. However, a combination in the genome of one and the same transgenic plant of a 1-SST encoding DNA sequence and a 1-FFT encoding DNA sequence, wherein either or both of said sequences are part of a recombinant construct, which combination is ensuring via the respective enzymes the production of inulin with a modified inulin profile (the host plant being an inulin or a non-inulin producing plant), has not been disclosed yet.
PCT patent application WO 96/21023 discloses a 1-SST encoding DNA sequence and a 1-FFT encoding DNA sequence both from J. artichoke, designated sst103 (or pSST103 when referring to the original clone being the sst103 inserted in the pBluescript SK vector) and fft111 (or pFFT111 when referring to the original clone being the fft111 inserted in the pBluescript SK vector), respectively, the construction of recombinant genes comprising said 1-SST enzyme or 1-FFT enzyme encoding sequence, and the transformation of plants (petunia and potato plants) by insertion of said sst103 gene or fft111 gene in the plant genome. Expression of the sst 103 gene in the transgenic plants was shown by analysis of the carbohydrate composition of the plants, revealing the presence of fructo-oligosaccharides with a DP up to 5. Expression of the fft111 gene in the transgenic plants was demonstrated in vitro on the basis of the ability of an extract of the transgenic plant to catalyse the synthesis of a polysaccharide Gxe2x88x92(F)n (n greater than 4) at the expense of submitted Gxe2x88x92(F)4 (G=glucosyl; F=fructosyl). No fructans were formed in the latter plants because the FFT enzyme needs fructo-oligosaccharides (such as FOS with DP of 3 or 4) for the synthesis of oligofructans and fructans with a higher DP. Based on the above, the patent application claims a method for producing a transgenic plant showing a modified inulin profile. A combination of DNA constructs comprising the 1-SST encoding DNA sequence, respectively the 1-FFT encoding DNA sequence, in one and the same transgenic plant has not been disclosed.
In food, feed, drinks and non-food applications, as well as for the manufacture of fructo-oligosaccharides, fructose and various derivatives of inulin, industry is increasingly making use of inulin. As a result thereof industry is continuously confronted with problems regarding the supply of inulin, particularly the supply of inulin compositions with desirable inulin profiles of highly linear polysaccharide chains. Said inulin compositions should preferably be readily and directly producible at industrial scale from plant sources at economically interesting costs. Preferably said production should be possible by conventional manufacturing processes and without additional process steps to modify the inulin profile of the native inulin, since each additional process step would inevitably increase the manufacturing costs and reduce the overall yield of suitable inulin.
The object of the present invention is, inter alia, to provide a method by which said and other problems can be solved, as well as to provide means for use in said method.
As indicated above, in WO 96/21023 a 1-SST encoding DNA sequence, termed sst103 , from the genome of J. artichoke is disclosed which codes for a 1-SST that catalyses in a plant the conversion of sucrose into kestose, thus enabling the plant to synthesise kestose (GFF), the smallest inulin molecule, from sucrose.
The inventors have now discovered the existence of a further 1-SST encoding DNA sequence in the genome of J. artichoke which apparently is part of a sleeping gene of said genome. The sequence has been identified in the form of its cDNA by DNA sequencing and this cDNA sequence, termed a33, is given in SEQ ID NO: 1 and is shown in FIG. 1 as A33. In FIG. 1 also the corresponding amino acid sequence is indicated, given in SEQ ID NO: 2.
Furthermore, the inventors have been able to identify a 1-FFT encoding DNA sequence in the genome of chicory and to identify its DNA sequence in the form of its cDNA by DNA sequencing. This cDNA sequence termed c86b, is given in SEQ ID NO: 3 and is shown in FIG. 2A as C86B, together with the corresponding amino acid sequence, given in SEQ ID NO: 4.
The inventors have found that the a33 cDNA sequence can be expressed in a host organism, particularly a plant or a plant part, to produce active 1-SST, when it is inserted in reading frame in a proper DNA construct in the genome of the organism. Said construct typically comprises the a33 cDNA sequence operably linked in the normal orientation to a promoter sequence and to a terminator sequence which are active in said host organism. When the host organism is a plant, the construct is preferably further comprising an operably linked DNA sequence encoding a targeting signal or a transit peptide which ensures targeting of the a33 encoded 1-SST enzyme to a specific subcellular compartment. The constructs, thus constituting a 1-SST enzyme encoding recombinant gene (in short herein 1-SST a33 gene), can be introduced into the genome of the host organism by conventional techniques.
Besides, the inventors have surprisingly found that said a33 cDNA sequence codes for an 1-SST enzyme which catalyses not only the transformation of sucrose to kestose and to lower oligofructoses (DPxe2x89xa6about 5), as do the known 1-SST enzyme encoding DNA sequences such as e.g. sst103 from J. artichoke, but encodes an 1-SST enzyme which is also able to catalyse the synthesis of higher fructo-oligosaccharides (DP up to about 10) in a plant. The a33 cDNA thus encodes in fact a 1-SST enzyme which has an activity which extends beyond the one of the known 1-SST enzymes, e.g. the 1-SST enzyme encoded by the sst103 cDNA, and has also an activity which is with respect to the building up of inulin chains functionally comparable to an aspect of a 1-FFT enzyme.
The inventors have also found that the c86b cDNA sequence can be expressed in a host organism, particularly a plant or a plant part, to produce active 1-FFT, when it is inserted in reading frame in a proper DNA construct in the genome of the host organism. Said construct typically comprises the c86b cDNA sequence operably linked in the normal orientation to a promoter sequence and to a terminator sequence which are active in said host organism. When the host organism is a plant, the construct is preferably further comprising an operably linked DNA sequence encoding a targeting signal or a transit peptide which ensures targeting of the c86b encoded 1-FFT enzyme to a specific subcellular compartment. The constructs, thus constituting a 1-FFT enzyme encoding recombinant gene (in short herein 1-FFT86b gene), can be introduced into the genome of the host organism by conventional techniques. Expression of said recombinant gene in a host plant results in the production of 1-FFT, which catalyses, as mentioned above, the stepwise synthesis of inulin by transformation of an oligofructose chain or inulin chain into an inulin molecule with a higher DP.
Furthermore, the inventors have found that a host organism, in particular a plant, can be transformed to comprise in its genome a combination of one or more expressible 1-SST enzyme encoding genes and one or more expressible 1-FFT enzyme encoding genes, wherein either the 1-SST enzyme encoding genes or the 1-FFT enzyme encoding genes or both comprise one or more recombinant genes containing one or more 1-SST enzyme encoding DNA sequences, respectively one or more 1-FFT enzyme encoding DNA sequences, of plant origin, resulting in a transgenic organism, particularly a transgenic plant, with a modified inulin producing profile.
Moreover, the inventors have found that the inulin producing profile of a plant and the profile of the inulin produced by a plant can be modified and even controlled, i.e. modified to yield a desired inulin profile, by producing a transgenic plant which comprises in its genome a combination of one or more expressible 1-SST enzyme encoding genes and one or more expressible 1-FFT enzyme encoding genes which code for 1-SST and 1-FFT enzymes with different kinetic properties. Either the 1-SST enzyme encoding genes or the 1-FFT enzyme encoding genes or both comprise one or more recombinant genes containing one or more 1-SST enzyme encoding DNA sequences, respectively one or more 1-FFT enzyme encoding DNA sequences, from plant sources, the latter 1-SST encoding sequences and 1-FFT encoding sequences being from the same or from different plant sources.
On the basis of said findings, the inventors have been able to provide a solution to the above mentioned and other problems by the present invention.
Accordingly, in a first embodiment, the invention relates to a method for producing a transgenic plant by transforming a host plant to comprise in its genome a combination of one or more expressible 1-SST enzyme encoding genes and one or more expressible 1-FFTenzyme encoding genes, wherein either the 1-SST enzyme encoding genes or the 1-FFT enzyme encoding genes or both comprise one or more expressible recombinant genes containing one or more expressible 1-SST enzyme encoding DNA sequences, respectively one or more expressible 1-FFT enzyme encoding DNA sequences which are of plant origin, comprising transforming a host plant by inserting one or more of said 1-SST enzyme encoding genes and/or one or more of said 1-FFT enzyme encoding genes into the genome of the host plant, yielding a transgenic plant with a modified inulin producing profile.
In accordance with the present invention, said 1-SST encoding DNA sequence(s) and said 1-FFT encoding DNA sequence(s) of said recombinant genes are not restricted to the sequences as obtained from the plant sources, but also include expressible homologous sequences thereof with a degree of homology of at least 70%, preferably at least 75%, more preferably at least 80% even more preferably at least 85%, and most preferably at least 90%, respectively, irrespective whether or not the homologous sequences are derived from plant sources or are obtained by mutagenesis of DNA sequences from plant sources or from micro-organisms.
By inulin is meant herein fructans of the inulin class, i.e. molecules of the general formulae GFn and Fm, as defined above, exclusive of fructans of the inulin neoseries class corresponding to the general formula
xe2x80x83F2xe2x88x92(1F2)mxe2x80x2xe2x88x926G1xe2x88x92(2F1)nxe2x80x2xe2x88x922F
wherein F and G respectively represent fructose and glucose, and mxe2x80x2 and nxe2x80x2 represent integers which can be the same or different.
By modified inulin producing profile is meant the production of inulin by a transgenic plant which is quantitatively and/or qualitatively different from the production of inulin by the non-transformed host plant.
By modified inulin profile is meant an inulin profile which is qualitatively different from the one of the inulin produced by the host plant, i.e. an inulin composition wherein the ratio of monosaccharides, disaccharides, oligo-saccharides and/or the distribution pattern of the chain length of the individual inulin molecules, i.e. the DP and the ({overscore (DP)}), are different from the ones of the inulin composition produced by the non-transformed host plant. For the sake of convenience, the term modified inulin producing profile is embracing herein a modified inulin producing profile, a controlled inulin producing profile as well as a modified inulin profile and a controlled inulin profile.
The non-transformed host plant suitable for the invention can be an inulin producing plant containing in its genome one or more 1-SST encoding genes and 1-FFT encoding genes or only 1-SST encoding genes, or a non-inulin producing plant.
If in the genome of the host plant a 1-FFT encoding gene and/or a 1-SST encoding gene is present, when producing the desired transgenic plant according to the present invention said gene or genes can be maintained or their expression can be totally or partially suppressed by known techniques. For example the expression of said genes can be suppressed through anti-sense expression or co-suppression strategies.
The said 1-SST enzyme encoding genes, respectively the said 1-FFT enzyme encoding genes, of the genome of the transgenic plant in accordance with the present invention, can consist of a native gene or a mixture of different native genes, of a mixture of native and recombinant genes, or of one or more different recombinant genes.
If said combination of 1-SST encoding gene(s) and 1-FFT encoding gene(s) comprises known 1-SST encoding gene(s), respectively known 1-FFT encoding gene(s), these known genes may be the ones which are present in the genome of the non-transformed host plant.
If said combination comprises recombinant genes, their 1-SST enzyme encoding sequence, respectively the 1-FFT enzyme encoding sequence, can be a known one or a novel one, from a plant source, or a homologous sequence thereof, as defined above, which encodes a 1-SST enzyme, respectively a 1-FFT enzyme.
If both the 1-SST enzyme encoding genes and the 1-FFT enzyme encoding genes comprise a recombinant gene, the 1-SST enzyme encoding sequence(s) and the 1-FFT enzyme encoding sequence(s) of said recombinant genes can be from the same or from different plant species.
If more than one 1-SST enzyme encoding DNA sequence, respectively more than one 1-FFT enzyme encoding DNA sequence, is present in the genome of the transgenic plant, the 1-SST encoding sequences, respectively the 1-FFT encoding sequences, may be identical or not, may be present in one or in different genes, and these 1-SST encoding genes, respectively 1-FFT encoding genes, may be present on one or on different chromosomes.
In a preferred embodiment of the invention, the recombinant 1-SST encoding gene comprises said a33 cDNA sequence or an expressible homologous sequence thereof as defined above.
In another preferred embodiment, the recombinant 1-FFT encoding gene comprises said c86b cDNA sequence or an expressible homologous sequence thereof as defined above.
In a further preferred embodiment, the recombinant 1-SST encoding gene comprises said a33 cDNA sequence or said homologous sequence and the 1-FFT encoding gene comprises said c86b cDNA sequence or said homologous sequence.
Said homologous sequences are at least 70% identical to said a33 cDNA, respectively to said c86b cDNA, irrespective of whether or not the homologous sequences are derived from another plant species or are obtained by mutagenesis of fructosyltransferase-encoding sequences from plant sources or from micro-organisms. Preferably the degree of homology is at least 75%, more preferably at least 80%, and even more preferably at least 85%. Most preferably the degree of homology is at least 90%.
In a further preferred embodiment, the transgenic plant has been produced by inserting into the genome of a host plant a combination of one or more genes with a known 1-FFT enzyme encoding DNA sequence and one or more genes with the 1-SST enzyme encoding a33 cDNA sequence or said respective homologous sequences thereof which encode a 1-FFT enzyme, respectively, a 1-SST enzyme. In another preferred embodiment, the transgenic plant has been produced by inserting into the genome of a host plant a combination of one or more genes with a known 1-SST encoding DNA sequence and one or more genes with the 1-FFT enzyme encoding c86b cDNA, or said respective homologous sequences thereof. In a further preferred embodiment, the transgenic plant has been produced by inserting into the genome of a host plant a combination of one or more genes with the 1-SST enzyme encoding a33 cDNA sequence or a said homologous sequence thereof and one or more genes with the 1-FFT enzyme encoding c86b cDNA or a said homologous sequence thereof. In a still further preferred embodiment of said execution forms of the invention, the host plant is a non-inulin producing plant.
Typical combinations of 1-SST encoding genes and 1-FFT encoding genes according to the invention comprise respectively a 1-SST enzyme encoding sequence or a said homologous sequence thereof and a 1-FFT enzyme encoding sequence or a said homologous sequence thereof selected from plant species of the Asteraceae family, with the 1-SST encoding sequence and the 1-FFT encoding sequence being selected from the same or from different plant species.
Further typical combinations of 1-SST encoding genes and 1-FFT encoding genes according to the invention comprise respectively a 1-SST enzyme encoding sequence or a said homologous sequence thereof and a 1-FFT enzyme encoding sequence or a said homologous sequence thereof selected from plant species from the same or different plant families of the group consisting of the Asteraceae (Compositae) and Campanulaceae, comprising plant species such as, for example, Echinops, species, Helianthus tuberosus, Cichorium intybus, Dahlia species, Cynara species, Viguiera species, such as e.g. Viguiera discolor, Viguiera deltoida, Viguiera annua, Viguiera lanata, Viguiera multiflora, Veronia herbacea, Scorzonera hispanica, Tragopogon porriflorus, Taraxacum, species, Arctium lappa, Campanula rapuncoloides and Bellis perennis. 
Typically suitable DNA sequences include, for example, the 1-SST encoding sequences sst103 (J. artichoke), a33 (J. artichoke), c33 (chicory), Genbank accession No U81520 (chicory), Genbank accession No Y09662 (Cynara scolymus), and the 1-FFT encoding sequences c86b (chicory) and fft111 (J. artichoke).
By the selection of a proper combination of one or more of said expressible 1-SST encoding genes and one or more of said expressible 1-FFT encoding genes, in combination with the selection of a proper ratio between the expression of the 1-SST encoding and 1-FFT encoding genes and the selection of a suitable host plant, a transgenic plant can be produced according to the invention with a desired modified inulin producing profile. In fact, the selection of proper combinations of said parameters by routine experiments, makes it possible to produce inulin with an almost tailored profile and to modify in a desired manner the inulin producing profile of a given host plant.
In a preferred embodiment, the genome of the transgenic plant comprises a combination of said expressible 1-SST encoding genes and said expressible 1-FFT encoding genes which induces the synthesis of native inulin with a degree of polymerisation which is higher, respectively lower, than the one of the native inulin produced, if any, by the non-transformed host plant.
When an inulin essentially composed of fructo-oligosaccharides with inulin chains with a DPxe2x89xa6about 10 is desired, a method is provided according to a particular embodiment of the present invention, for producing a transgenic plant which produces inulin with such profile. This is very advantageous because no conventional, partial hydrolysis step of inulin with longer carbohydrate chains, such as e.g. standard grade chicory inulin with a ({overscore (DP)}) of about 10, needs to be included in the production process of said inulo-oligosaccharides. Accordingly, in a first variant, a method is provided for producing a transgenic plant by transforming a host plant to comprise in its genome a combination of one or more expressible 1-SST encoding genes, originating from the host plant or from a different plant source and one or more 1-SSTa33 genes. If the host plant contains a native 1-SST encoding gene and a native 1-FFT encoding gene in its genome, the expression of the native 1-FFT encoding gene or of both the native 1-SST encoding gene and the native 1-FFT encoding gene can optionally be suppressed by known techniques, for example through anti-sense expression. In a second variant, a transgenic plant is produced by inserting into the genome of a host plant which does not contain a 1-SST encoding gene nor a 1-FFT encoding gene, one or more 1-SST a33 genes or genes which contain a cDNA sequence which is an homologous sequence thereof, as defined above, which encodes an a33 1-SST enzyme. This particular embodiment of the present invention has become possible as a result of the fact that the 1-SST a33 gene codes for an enzyme which presents an activity of a 1-SST enzyme, i.e. catalysing the synthesis of kestose from sucrose, but also presents a moderate activity with respect to the building up of inulin chains which is comparable to an aspect of a 1-FFT enzyme activity, i.e. catalysing the synthesis of fructo-oligosaccharides from kestose or fructo-oligosaccharides with a low DP.
The selection of the optimal combination and ratio of the number of concerned 1-SST and 1-FFT enzyme encoding DNA sequences in combination with the selection of the most suitable plant species for the production of a desired inulin profile can be made by the skilled person according to conventional techniques, for example through routine experiments.
In the method according to the invention, the transgenic plant can be produced by inserting into the genome of the host plant by conventional techniques one or more expressible 1-SSTa33 genes or expressible homologues thereof, or one or more of said expressible 1-SST encoding genes from plant sources or said homologues thereof and/or one or more of said expressible 1-FFT encoding genes from plant sources or said expressible homologues thereof, resulting in a transgenic plant which comprises in its genome one or more 1-SSTa33 enzyme encoding genes or a combination of said 1-SST encoding genes and said 1-FFT encoding genes as defined herein above.
In a typical execution form, a cell of a host plant is transformed to comprise in its genome a said combination of 1-SST encoding genes and 1-FFT encoding genes as defined above, by inserting by conventional techniques one or more of said genes into the genome of the cell, followed by regenerating a transgenic plant from said transformed cell. If the host plant is transformed with both said 1-SST encoding genes and said 1-FFT encoding genes, the genes can be inserted into the host genome simultaneously or in subsequent steps.
In a typical execution form, said method comprises the following subsequent steps which can be carried out by conventional techniques:
i) the preparation of a recombinant gene construct comprising one or more 1-SST enzyme encoding DNA sequences, respectively 1-FFT enzyme encoding DNA sequences as defined above, operably linked to a promoter sequence active in said host plant and a terminator sequence active in said host plant,
ii) introduction of the recombinant gene construct obtained in step i) into the genome of a cell of the host plant, and
iii) regeneration of the transformed plant cell obtained in step ii) to the corresponding transgenic plant.
More specifically the method of the invention comprises preferably the following subsequent steps:
a) the construction of a recombinant gene, i.e. a recombinant DNA construct, comprising essentially the following sequences:
1. a promoter which ensures the formation of a functional RNA or a functional protein in the intended target plant, target organs, tissues or cells thereof,
2. one or more copies of a DNA sequence encoding respectively 1-SST or 1-FFT enzyme, functionally connected to said promoter,
3. a transcription terminator operationally connected to said 1-SST or 1-FFT enzyme encoding DNA sequence,
4. a DNA sequence encoding a targeting signal or a transit peptide which ensures targeting of the 1-SST enzyme, respectively the 1-FFT enzyme to a specific subcellular compartment,
b) the introduction of the recombinant gene obtained in a) into the genome of the host plant yielding genetically modified material, typically a cell, comprising said combination of 1-SST enzyme and 1-FFT enzyme encoding sequences, and regeneration of the genetically transformed material in the corresponding transformed host plant.
In the recombinant DNA construct according to the present invention, one or more copies of the 1-SST and 1-FFT enzyme encoding DNA sequences are preferably linked to one or more regulatory sequences which are operational in the host plant ensuring proper expression of said DNA sequence at a sufficiently high expression level in the host plant, in the different plant organs, tissues or cells. Regulatory sequences are, for example, a promoter, a termination signal and a transcription or translational enhancer. A promoter can, for example, be the 35S promoter of the cauliflower mosaic virus (CaMV) or an organ-specific promoter like the tuber-specific potato proteinase inhibitor II promoter, or any other inducible or tissue-specific promoter.
The production of inulin being particularly advantageous in organs storing large amounts of sucrose, such as the tap roots of sugar beet or the stems of sugar cane, a highly preferred promoter is a promoter which is active in organs and cell types which normally accumulate sucrose (the primary substrate for inulin synthesis).
The production of inulin is particularly advantageous in the vacuole which can accumulate very high concentrations of sucrose (e.g. up to about 500 mol mxe2x88x923). Accordingly, in the recombinant DNA according to the present invention, the DNA sequence encoding 1-SST or 1-FFT enzyme is preferably linked to a sequence encoding a transit peptide which directs the mature 1-SST or 1-FFT enzyme protein to a subcellular compartment containing sucrose, such as for example said vacuole.
In a preferred embodiment, the host plant is a non-inulin producing plant; in another preferred embodiment, the host plant is an inulin producing plant.
Typical host plants for use in the method according to the invention are plants which can easily be grown, which are rather resistant to attack by injurious organisms such as insects and fungi, which give a high yield of plant material per hectare and which can be easily harvested and processed. Besides, said plants should after transformation be able to produce large amounts of inulin, give a high content of produced inulin based on fresh plant material and preferably be able to deposit said inulin in a concentrated manner in parts of the plant, preferably in tap roots or tubers, which can be easily harvested, stored and processed.
Other typical host plants for use in the method of the invention are non-inulin producing plants which are quite sensitive to abiotic stresses such as, for example, drought, cold, and other ones. Transformation of said host plants according to the present invention resulting in a transgenic plant which is producing inulin, even in a quite low level, may significantly increase the resistance of the plant against said abiotic stresses, particularly against drought and/or cold.
Typical host plants suitable for use in the method according to the invention include corn, wheat, rice, barley, sorghum, millets, sunflower, cassava, canola, soybean, oil palm, groundnut, cotton, sugar cane, chicory, bean, pea, cow pea, banana, tomato, beet, sugar beet, Jerusalem artichoke, tobacco, potato, sweet potato, coffee, Gocoa and tea.
In a further embodiment, the present invention provides a method for modifying the inulin producing profile of a plant and for controlling the profile of the inulin produced by a plant, which comprises genetically transforming a plant by inserting into its genome one or more expressible 1-SSTa33 genes or expressible homologues thereof, or one or more expressible 1-SST encoding genes, and/or one or more expressible 1-FFT encoding genes, as defined above, yielding a transgenic plant comprising in its genome a combination of said 1-SST encoding genes and said 1-FFT encoding genes, as defined herein above, which plant, when cultured under conventional conditions, shows a modified inulin producing profile.
In still another embodiment, the present invention relates to a method for producing inulin from plant material, particularly inulin with a modified profile, by conventional techniques, wherein the source plant material for the method is material, typically tap roots or tubers, from a transgenic plant which comprises in its genome one or more expressible 1-SST a33 enzyme encoding genes, or a combination of one or more expressible 1-SST encoding genes and one or more expressible 1-FFT encoding genes, or expressible homologues of said genes, as defined above. Such transgenic plant is obtainable by a method of the present invention as described herein before.
The production of inulin from plant parts is well known in the art and commonly comprises (i) an isolation step, wherein the crude, native inulin is isolated from the plant material (typically involving extraction of shredded plant parts e.g. tap roots or tubers, with warm water ), followed by (ii) a purification step, typically comprising a depuration treatment (involving liming and carbonatation or another flocculation technique and filtration) followed by a refining treatment (involving treatment over ion-exchangers, treatment with active carbon and filtration) and optionally a concentration of the purified inulin solution, and (iii) an isolation step wherein the inulin is isolated in particulate form from the purified inulin solution obtained in (ii), for example by spray-drying or by directed crystallisation, filtration and drying.
The invention further embraces the novel DNA sequences a33 cDNA and c86b cDNA, and homologous sequences thereof, as defined above, encoding respectively 1-SST enzyme and 1-FFT enzyme, as well as novel recombinant DNA constructs, novel recombinant genes and vectors comprising one or more of said novel cDNA sequences. Said novel cDNA sequences are suitable intermediates for the construction of said novel recombinant DNA constructs and recombinant genes, which are in turn, suitable intermediates and tools for the production of transgenic plants presenting a modified inulin producing profile according to the present invention.
The invention furthermore embraces a novel combination of one or more expressible 1-SST encoding genes and one or more expressible 1-FFT encoding genes, wherein either the 1-SST encoding genes or the 1-FFT encoding genes or both comprise one or more recombinant genes of which one or more of the 1-SST enzyme encoding DNA sequences and one or more of the 1-FFT enzyme encoding DNA sequences are of plant origin or homologues thereof, as defined above. Said novel combination of genes constitutes a suitable intermediate and tool for the production of transgenic plants according to the present invention.
The invention also embraces a transgenic plant, producible by a method according to the present invention, the genome of which comprises a combination of one or more expressible 1-SST encoding genes and one or more expressible 1-FFT encoding genes, as defined above.
The present invention also includes cells, plant tissue, plant parts, roots and shoots of transgenic plants according to the invention as well as seeds thereof, which comprise in their genome a combination of one or more expressible 1-SST encoding genes and one or more expressible 1-FFT encoding genes, as defined above.
The present invention also includes novel inulin compositions, i.e. inulin having a novel inulin profile, obtainable by a method according to the present invention, and the use thereof in the manufacture of food, feed, drinks, and non-food compositions, of derivatives of inulin , and of partial and complete hydrolysates of inulin.
In still a further embodiment, the present invention relates to a purified and isolated polypeptide having an amino acid sequence as shown in SEQ ID NO:2, respectively in SEQ ID NO: 4, and to purified and isolated respective homologues thereof having at least 70% homology, preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, and most preferably at least 90% homology, as well as to a fragment of said polypeptides, which polypeptides, said homologues thereof and said fragments thereof have 1-SST, respectively 1-FFT, activity.
Said polypeptides, homologues and fragments can be obtained according to conventional techniques. For example the polypeptides of SEQ ID NO: 2 and of SEQ ID NO: 4 can be obtained from chicory roots through a purification procedure based on ammonium sulphate precipitation, followed by lectin affinity chromatography, anion- and cation exchange chromatography.
In an ultimate embodiment, the present invention relates to antibodies capable of specifically binding one or more polypeptides and/or homologues and/or fragments thereof as defined above.